Abstract /Summary: Streptococcus pneumoniae, a principal cause of pneumonia and otitis media, escapes from vaccine protection and antibiotic therapy by acquiring new genes through genetic transformation from other strains or species. Genetic transformation in S. pneumoniae (pneumococcus) is regulated by a quorum sensing system that induces the expression of its sole alternative sigma factor and master regulator of competence, SigX, as well as expression of a second regulator of competence, ComW. Together, the two proteins, SigX and ComW, direct transcription of numerous transformation-specific genes. SigX directly binds to core RNA polymerase, resulting in a transient and global shift in gene expression; however we have not yet been able to assign a direct function to ComW. Previous experimental data suggest that ComW may act at the point of sigma factor switching between SigA and SigX at the onset of competence. As the interplay between SigX and ComW represents a potentially unique sigma-factor switching mechanism and is required for genetic transformation, we aim to determine the role of ComW and to dissect the molecular interaction between ComX and ComW. Not only is the biochemical function of ComW currently unknown, but the lack of known homologs suggests a novel fold and mechanism of action. To study this system, we will take a biochemical and biophysical approach to characterize ComW both functionally and structurally, probe its interaction with SigX in vitro, and support our observations in vivo by genetic analysis directly in S. pneumoniae. Medical Relevance. Most pathogenic streptococci of the mitis and anginosus groups share this mechanism of control of natural genetic transformation. Genetic transformation is an important path for genetic flexibility in pneumococcus, where it is documented as key to vaccine escape and creation and spread of drug-resistance genes. Because Streptococcus pneumoniae is a model organism for the study of DNA uptake, this work on the mechanism that connects quorum sensing to natural competence will have broad impact on understanding and targeting the many similar peptide signaling systems among Gram positive bacteria that are often associated with the ability of these bacteria to cause disease.